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Talimogene Laherparepvec (OncoVEXmGMCSF), an oncolytic virus, immune checkpoint inhibitor anti-programmed cell death protein 1 (anti-PD1), and BRAF inhibition (BRAFi), are all clinically approved for treatment of melanoma patients and are effective through diverse mechanisms of action. Individually, these therapies also have an effect on the tumor immune microenvironment (TIME). Evaluating the combination effect of these three therapies on the TIME can help determine when combination therapy is most appropriate for further study. In this study, we use a transgenic murine melanoma model (Tyr::CreER; BRAFCA/+; PTENflox/flox), to evaluate the TIME in response to combinations of BRAFi, anti-PD1, and OncoVEXmGMCSF. We find that mice treated with the triple combination BRAFi + anti-PD1 + OncoVEXmGMCSF have decreased tumor growth compared to BRAFi alone and prolonged survival compared to control. Flow cytometry shows an increase in percent CD8 + /CD3 + cytotoxic T Lymphocytes (CTLs) and a decrease in percent FOXP3 + /CD4 + T regulatory cells (Tregs) in tumors treated with OncoVEXmGMCSF compared to mice not treated with OncoVEXmGMCSF. Immunogenomic analysis at 30d post-treatment shows an increase in Th1 and interferon-related genes in mice receiving OncoVEXmGMCSF + BRAFi. In summary, treatment with combination BRAFi + anti-PD1 + OncoVEXmGMCSF is more effective than any single treatment in controlling tumor growth, and groups receiving OncoVEXmGMCSF had more tumoral infiltration of CTLs and less intratumoral Tregs in the TIME. This study provides rational basis to combine targeted agents, oncolytic viral therapy, and checkpoint inhibitors in the treatment of melanoma. [ABSTRACT FROM AUTHOR]

Background: Immunotherapy, specifically immune checkpoint inhibitors (ICIs), including anti‐programmed cell death 1 (anti‐PD1), has recently received clinical approval for the treatment of adult hepatocellular carcinoma (HCC). However, the safety and efficacy of ICIs prior to solid organ transplant are unknown, especially in pediatrics. Safety reports are variable in adults, with some series describing subsequent allograft rejection and loss while others report successful transplants without allograft rejection.As ICIs stimulate the immune system by blocking the interaction between PD1 and the ligand‐receptor pair programmed cell death‐ligand 1 (PDL1), the downstream effects of T‐cell activation increase the risk of graft rejection. Methods: Here, we present a case of an adolescent with moderately differentiated non‐fibrolamellar HCC treated with pembrolizumab, an anti‐PD1 therapy, who subsequently underwent successful orthotopic liver transplantation (OLT). Results: Our patient received an OLT 138 days from the last pembrolizumab dose with graft preservation. The patient has no evidence of recurrent disease or any episode of allograft rejection 48 months post OLT. Staining of tumor and normal tissues from longitudinal specimens finds PDL1 positive Kupffer cells present in normal liver and peritumoral areas with no changes post anti‐PD1 therapy. In contrast, tumor cells were negative for PDL1. Conclusion: This case represents a basis for optimism in potential use of anti‐PD1 therapy in liver transplant candidates and supports further investigation of immune checkpoint inhibitors use in this unique patient population. [ABSTRACT FROM AUTHOR]

Eribulin inhibits microtubule polymerization and suppresses epithelial-mesenchymal transition. Conventional pathology approaches have not identified a precise predictive biomarker for Eribulin. We performed qmIF on pre-treatment tissue from 11 patients (6 TNBC, 5 HGSOC) treated with Eribulin-LF. T-lymphocytes were the dominant immune-subset in TME, with higher levels detected in stroma vs tumor (9% vs 2%). Greater density of CD3+ (p = 0.01) and CD3 + CD8+ (p = 0.03) cells and closer proximity between CD3 + CD8+ and tumor cells was observed in the patients with disease control (PR + SD) vs. progressive disease. QmIF identified an association between TIL infiltration and Eribulin-LF sensitivity, which should be evaluated further in prospective studies. [ABSTRACT FROM AUTHOR]

The presence of actinic keratoses (AKs) increases a patient's risk of developing squamous cell carcinoma by greater than six-fold. We evaluated the effect of topical treatment with imiquimod on the tumor microenvironment by measuring transcriptomic differences in AKs before and after treatment with imiquimod 3.75%. Biopsies were collected prospectively from 21 patients and examined histologically. RNA was extracted and transcriptomic analyses of 788 genes were performed using the nanoString assay. Imiquimod decreased number of AKs by study endpoint at week 14 (p < 0.0001). Post-imiquimod therapy, levels of CDK1, CXCL13, IL1B, GADPH, TTK, ILF3, EWSR1, BIRC5, PLAUR, ISG20, and C1QBP were significantly lower (adjusted p < 0.05). Complete responders (CR) exhibited a distinct pattern of inflammatory gene expression pre-treatment relative to incomplete responders (IR), with alterations in 15 inflammatory pathways (p < 0.05) reflecting differential expression of 103 genes (p < 0.05). Presence of adverse effects was associated with improved treatment response. Differences in gene expression were found between pre-treatment samples in CR versus IR, suggesting that higher levels of inflammation pre-treament may play a part in regression of AKs. Further characterization of the immune micro-environment in AKs may help develop biomarkers predictive of response to topical immune modulators and may guide therapy. [ABSTRACT FROM AUTHOR]

Purpose: To compare the prognostic value of imaging biomarkers derived from a quantitative analysis of baseline 18F-FDG-PET/CT in patients with mucosal melanoma (Muc-M) or cutaneous melanoma (Cut-M) treated with immune checkpoint inhibitors (ICIs). Methods: In this retrospective monocentric study, we included 56 patients with non-resectable Muc-M (n = 24) or Cut-M (n = 32) who underwent baseline 18F-FDG-PET/CT before treatment with ICIs between 2011 and 2017. Parameters were extracted from (i) tumoral tissues: SUVmax, SUVmean, TMTV (total metabolic tumor volume), and TLG (total lesion glycolysis) and (ii) lymphoid tissues: BLR (bone marrow-to-liver SUVmax ratio) and SLR (spleen-to-liver SUVmax ratio). Association with survival and response was evaluated using Cox prediction models, Student's t tests, and Spearman's correlation respectively. p < 0.05 was considered significant. Results: Majority of ICIs were anti-PD1 (92.9%, n = 52/56). All 18F-FDG-PET/CT were positive. Overall (Muc-M to Cut-M), ORR was 33%:42%, DCR was 56%:69%, median follow-up was 25.0:28.9 months, median PFS was 4.7:10.7 months, and median OS was 23.9:28.3 months. In Muc-M, increased tumor SUVmax was associated with shorter OS while it was not correlated with PFS, ORR, or DCR. In Cut-M, increased TMTV and increased BLR were independently associated with shorter OS, shorter PFS, and lower response (ORR, DCR). Conclusion: While all Muc-M and Cut-M were FDG avid, prognostic imaging biomarkers differed. For Muc-M patients treated with ICI, the only prognostic imaging biomarker was a high baseline maximal glycolytic activity (SUVmax), whereas for Cut-M patients, baseline metabolic tumor burden or bone marrow metabolism was negatively correlated to ICI response duration. [ABSTRACT FROM AUTHOR]

Immunotherapy has revolutionized the treatment of melanoma. Targeting of the immune checkpoints cytotoxic T-lymphocyte-associated protein 4 and programmed cell death protein 1 has led to improved survival in a subset of patients. Unfortunately, the use of immune checkpoint inhibitors is associated with significant side effects and many patients do not respond to treatment. Thus, there is an urgent need both for prognostic biomarkers to estimate risk and for predictive biomarkers to determine which patients are likely to respond to therapy. In this review, prognostic and predictive biomarkers that are an active area of research are outlined. Of note, certain transcriptomic signatures are already used in the clinic, albeit not routinely, to prognosticate patients. In the predictive setting, programmed cell death protein ligand 1 expression has been shown to correlate with benefit but is not precise enough to be used as an exclusionary biomarker. Future investigation will need to focus on biomarkers that are easily reproducible, cost effective, and accurate. The use of readily available clinical material, such as serum or hematoxylin and eosin-stained images, may offer one such path forward. [ABSTRACT FROM AUTHOR]

Purpose: To enhance clinician's decision-making by diagnosing hepatocellular carcinoma (HCC) in cirrhotic patients with indeterminate liver nodules using quantitative imaging features extracted from triphasic CT scans.Material and Methods: We retrospectively analyzed 178 cirrhotic patients from 27 institutions, with biopsy-proven liver nodules classified as indeterminate using the European Association for the Study of the Liver (EASL) guidelines. Patients were randomly assigned to a discovery cohort (142 patients (pts.)) and a validation cohort (36 pts.). Each liver nodule was segmented on each phase of triphasic CT scans, and 13,920 quantitative imaging features (12 sets of 1160 features each reflecting the phenotype at one single phase or its change between two phases) were extracted. Using machine-learning techniques, the signature was trained and calibrated (discovery cohort), and validated (validation cohort) to classify liver nodules as HCC vs. non-HCC. Effects of segmentation and contrast enhancement quality were also evaluated.Results: Patients were predominantly male (88%) and CHILD A (65%). Biopsy was positive for HCC in 77% of patients. LI-RADS scores were not different between HCC and non-HCC patients. The signature included a single radiomics feature quantifying changes between arterial and portal venous phases: DeltaV-A_DWT1_LL_Variance-2D and reached area under the receiver operating characteristic curve (AUC) of 0.70 (95%CI 0.61-0.80) and 0.66 (95%CI 0.64-0.84) in discovery and validation cohorts, respectively. The signature was influenced neither by segmentation nor by contrast enhancement.Conclusion: A signature using a single feature was validated in a multicenter retrospective cohort to diagnose HCC in cirrhotic patients with indeterminate liver nodules. Artificial intelligence could enhance clinicians' decision by identifying a subgroup of patients with high HCC risk.Key Points: • In cirrhotic patients with visually indeterminate liver nodules, expert visual assessment using current guidelines cannot accurately differentiate HCC from differential diagnoses. Current clinical protocols do not entail biopsy due to procedural risks. Radiomics can be used to non-invasively diagnose HCC in cirrhotic patients with indeterminate liver nodules, which could be leveraged to optimize patient management. • Radiomics features contributing the most to a better characterization of visually indeterminate liver nodules include changes in nodule phenotype between arterial and portal venous phases: the "washout" pattern appraised visually using EASL and EASL guidelines. • A clinical decision algorithm using radiomics could be applied to reduce the rate of cirrhotic patients requiring liver biopsy (EASL guidelines) or wait-and-see strategy (AASLD guidelines) and therefore improve their management and outcome. [ABSTRACT FROM AUTHOR]

Background: Immune checkpoint inhibitors (ICIs) for solid tumors, including those targeting programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have shown impressive clinical efficacy, however, most patients do not achieve durable responses. One major therapeutic obstacle is the immunosuppressive tumor immune microenvironment (TIME). Thus, we hypothesized that a strategy combining tumor-directed radiation with TIME immunomodulation could improve ICI response rates in established solid tumors. Methods: Using a syngeneic mouse model of human papillomavirus (HPV)-associated head and neck cancer, mEER, we developed a maximally effective regimen combining PD-1 and CTLA-4 inhibition, tumor-directed radiation, and two existing immunomodulatory drugs: cyclophosphamide (CTX) and a small-molecule inducible nitric oxide synthase (iNOS) inhibitor, L-n6-(1-iminoethyl)-lysine (L-NIL). We compared the effects of the various combinations of this regimen on tumor growth, overall survival, establishment of immunologic memory, and immunologic changes with flow cytometry and quantitative multiplex immunofluorescence. Results: We found PD-1 and CTLA-4 blockade, and radiotherapy alone or in combination, incapable of clearing established tumors or reversing the unfavorable balance of effector to suppressor cells in the TIME. However, modulation of the TIME with cyclophosphamide (CTX) and L-NIL in combination with dual checkpoint inhibition and radiation led to rejection of over 70% of established mEER tumors and doubled median survival in the B16 melanoma model. Anti-tumor activity was CD8+ T cell-dependent and led to development of immunologic memory against tumor-associated HPV antigens. Immune profiling revealed that CTX/L-NIL induced remodeling of myeloid cell populations in the TIME and tumor-draining lymph node and drove subsequent activation and intratumoral infiltration of CD8+ effector T cells. Conclusions: Overall, this study demonstrates that modulation of the immunosuppressive TIME is required to unlock the benefits of ICIs and radiotherapy to induce immunologic rejection of treatment-refractory established solid tumors. [ABSTRACT FROM AUTHOR]

Background: Chemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects. Methods: Therefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes. Results: We show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8+ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8+ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner. Conclusions: Overall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation. [ABSTRACT FROM AUTHOR]

Purpose: An imaging-based stratification tool is needed to identify melanoma patients who will benefit from anti Programmed Death-1 antibody (anti-PD1). We aimed at identifying biomarkers for survival and response evaluated in lymphoid tissue metabolism in spleen and bone marrow before initiation of therapy. Methods: This retrospective study included 55 patients from two institutions who underwent 18F-FDG PET/CT before anti-PD1. Parameters extracted were SUVmax, SUVmean, HISUV (SUV-based Heterogeneity Index), TMTV (total metabolic tumor volume), TLG (total lesion glycolysis), BLR (Bone marrow-to-Liver SUVmax ratio), and SLR (Spleen-to-Liver SUVmax ratio). Each parameter was dichotomized using the median as a threshold. Association with survival, best overall response (BOR), and transcriptomic analyses (NanoString assay) were evaluated using Cox prediction models, Wilcoxon tests, and Spearman's correlation, respectively. Results: At 20.7 months median follow-up, 33 patients had responded, and 29 patients died. Median PFS and OS were 11.4 (95%CI 2.7–20.2) and 28.5 (95%CI 13.4–43.8) months. TMTV (>25cm3), SLR (>0.77), and BLR (>0.79) correlated with shorter survival. High TMTV (>25 cm3), SLR (>0.77), and BLR (>0.79) correlated with shorter survival, with TMTV (HR PFS 2.2, p = 0.02, and HR OS 2.5, p = 0.02) and BLR (HR OS 2.3, p = 0.04) remaining significant in a multivariable analysis. Low TMTV and TLG correlated with BOR (p = 0.03). Increased glucose metabolism in bone marrow (BLR) was associated with transcriptomic profiles including regulatory T cell markers (p < 0.05). Conclusion: Low tumor burden correlates with survival and objective response while hematopoietic tissue metabolism correlates inversely with survival. These biomarkers should be further evaluated for potential clinical application. [ABSTRACT FROM AUTHOR]

Purpose: To establish a novel preclinical model for stereotactic radiosurgery (SRS) with combined mouselike phantom quality assurance in the setting of brain metastases.Methods and Materials: C57B6 mice underwent intracranial injection of B16-F10 melanoma cells. T1-weighted postcontrast magnetic resonance imaging (MRI) was performed on day 11 after injection. The MRI images were fused with cone beam computed tomography (CBCT) images using the Small Animal Radiation Research Platform (SARRP). The gross tumor volume (GTV) was contoured using the MRI. A single sagittal arc using the 3 × 3 mm2 collimator was used to deliver 18 Gy prescribed to the isocenter. MRI was performed 7 days after radiation treatment, and the dose delivered to the mice was confirmed using 2 mouselike anthropomorphic phantoms: 1 in the axial orientation and the other in the sagittal orientation. The SARRP output was measured using a PTW Farmer type ionization chamber as per the American Association of Physicists in Medicine Task Group report 61, and the H-D curve was generated up to a maximum dose of 30 Gy. Irradiated films were analyzed based on optical density distribution and H-D curve.Results: The tumor volume on day 11, before intervention, was 2.48 ± 1.37 mm3 in the no-SRS arm versus 3.75 ± 1.19 mm3 in the SRS arm (NS). In the SRS arm, GTV maximum dose (Dmax) and mean dose were 2048 ± 207 and 1785 ± 14 cGy. Using the mouselike phantoms, the radiochromic film showed close precision in comparison with projected isodose lines, with a Dmax of 1903.4 and 1972.7 cGy, the axial and sagittal phantoms, respectively. Tumor volume 7 days after treatment was 7.34 ± 8.24 mm3 in the SRS arm and 60.20 ± 40.4 mm3 in the no-SRS arm (P=.009). No mice in the control group survived more than 22 days after implantation, with a median overall survival (mOS) of 19 days; mOS was not reached in the SRS group, with 1 death noted.Conclusions: Single-fraction SRS of 18 Gy delivered in a single arc can be delivered accurately with MRI T1-weighted postcontrast-based treatment planning. The mouse like phantom allows for verification of dose delivery and accuracy. [ABSTRACT FROM AUTHOR]

Melanoma immunotherapy has been an area of intense research for decades, and this work is now yielding more tangible results for patients. Work has focused on 4 main areas: cytokine therapy, administration of immune-modulating antibodies, adoptive T-cell therapy, and vaccines. Cytokine therapy is an established treatment for advanced melanoma, and immune-modulating antibodies have recently emerged as an exciting new area of drug development with efficacy now established in a phase III trial. Adoptive T-cell therapy provides the proof of principle that T cells can attack and eliminate tumors. It has been challenging, however, to adapt this treatment for widespread use. Vaccines have generally yielded poor results, but intratumor pathogen-based strategies have shown encouraging results in recent trials, perhaps due to stronger immune stimulation. A review of the field of melanoma immunotherapy is provided here, with emphasis on those agents that have reached clinical testing. Novel strategies to induce the immune system to attack melanomas are reviewed. In the future, it is envisioned that immunotherapy will have further application in combination with cytotoxic and targeted therapies. © 2010 Mount Sinai School of Medicine [ABSTRACT FROM AUTHOR]

Simple Summary: Melanoma constitutes a major public health risk, with the rates of diagnosis increasing on a yearly basis. Monitoring for risk factors and preventing dangerous behaviors that increase melanoma risk, such as tanning, are important measures for melanoma prevention. Additionally, assessing the effectiveness of various methods to prevent sun exposure and sunburns—which can lead to melanoma—is important to help identify ways to reduce the development of melanoma. We summarize the recent evidence regarding the heritable and behavioral risks underlying melanoma, as well as the current methods used to reduce the risk of developing melanoma and to improve the diagnosis of this disease. (1) Melanoma is the most aggressive dermatologic malignancy, with an estimated 106,110 new cases to be diagnosed in 2021. The annual incidence rates continue to climb, which underscores the critical importance of improving the methods to prevent this disease. The interventions to assist with melanoma prevention vary and typically include measures such as UV avoidance and the use of protective clothing, sunscreen, and other chemopreventive agents. However, the evidence is mixed surrounding the use of these and other interventions. This review discusses the heritable etiologies underlying melanoma development before delving into the data surrounding the preventive methods highlighted above. (2) A comprehensive literature review was performed to identify the clinical trials, observational studies, and meta-analyses pertinent to melanoma prevention and incidence. Online resources were queried to identify epidemiologic and clinical trial information. (3) Evidence exists to support population-wide screening programs, the proper use of sunscreen, and community-targeted measures in the prevention of melanoma. Clinical evidence for the majority of the proposed preventive chemotherapeutics is presently minimal but continues to evolve. (4) Further study of these chemotherapeutics, as well as improvement of techniques in artificial intelligence and imaging techniques for melanoma screening, is warranted for continued improvement of melanoma prevention. [ABSTRACT FROM AUTHOR]

Simple Summary: Immunotherapy has changed the landscape of treatment modalities available for many different types of malignancies. However, the factors that influence the success of immunotherapeutics have not been as clearly seen in advanced prostate cancer, likely due to immunosuppressive factors that exist within the prostate cancer tumor microenvironment. While there have been many immunotherapeutics used for prostate cancer, the majority have targeted a single immunosuppressive mechanism resulting in limited clinical efficacy. More recent research centered on elucidating the key mechanisms of immune resistance in the prostate tumor microenvironment has led to the discovery of a range of new treatment targets. With that in mind, many clinical trials have now set out to evaluate combination immunotherapeutic strategies in patients with advanced prostate cancer, in the hopes of circumventing the immunosuppressive mechanisms. The use of immunotherapy has become a critical treatment modality in many advanced cancers. However, immunotherapy in prostate cancer has not been met with similar success. Multiple interrelated mechanisms, such as low tumor mutational burden, immunosuppressive cells, and impaired cellular immunity, appear to subvert the immune system, creating an immunosuppressive tumor microenvironment and leading to lower treatment efficacy in advanced prostate cancer. The lethality of metastatic castrate-resistant prostate cancer is driven by the lack of therapeutic regimens capable of generating durable responses. Multiple strategies are currently being tested to overcome immune resistance including combining various classes of treatment modalities. Several completed and ongoing trials have shown that combining vaccines or checkpoint inhibitors with hormonal therapy, radiotherapy, antibody–drug conjugates, chimeric antigen receptor T cell therapy, or chemotherapy may enhance immune responses and induce long-lasting clinical responses without significant toxicity. Here, we review the current state of immunotherapy for prostate cancer, as well as tumor-specific mechanisms underlying therapeutic resistance, with a comprehensive look at the current preclinical and clinical immunotherapeutic strategies aimed at overcoming the immunosuppressive tumor microenvironment and impaired cellular immunity that have largely limited the utility of immunotherapy in advanced prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Immunotherapy, in particular checkpoint blockade, has changed the clinical landscape of metastatic melanoma. Nonetheless, the majority of patients will either be primary refractory or progress over follow up. Management of patients progressing on first-line immunotherapy remains challenging. Expanded treatment options with combination immunotherapy has demonstrated efficacy in patients previously unresponsive to single agent or alternative combination therapy. Case presentation: We describe the case of a patient with diffusely metastatic melanoma, including brain metastases, who, despite being treated with stereotactic radiosurgery and dual CTLA-4/PD-1 blockade (ipilimumab/nivolumab), developed systemic disease progression and innumerable brain metastases. This patient achieved a complete CNS response and partial systemic response with standard whole brain radiation therapy (WBRT) combined with Talimogene laherparepvec (T-Vec) and pembrolizumab. Conclusion: Patients who do not respond to one immunotherapy combination may respond during treatment with an alternate combination, even in the presence of multiple brain metastases. Biomarkers are needed to assist clinicians in evidence based clinical decision making after progression on first line immunotherapy to determine whether response can be achieved with second line immunotherapy. [ABSTRACT FROM AUTHOR]

Purpose: TPST-1120 is a first-in-class oral inhibitor of peroxisome proliferator-activated receptor α (PPARα), a fatty acid ligand-activated transcription factor that regulates genes involved in fatty acid oxidation, angiogenesis, and inflammation, and is a novel target for cancer therapy. TPST-1120 displayed antitumor activity in xenograft models and synergistic tumor reduction in syngeneic tumor models when combined with anti-PD-1 agents., Experimental Design: This phase I, open-label, dose-escalation study (NCT03829436) evaluated TPST-1120 as monotherapy in patients with advanced solid tumors and in combination with nivolumab in patients with renal cell carcinoma (RCC), cholangiocarcinoma (CCA), or hepatocellular carcinoma. Objectives included evaluation of safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity (RECIST v1.1)., Results: A total of 39 patients enrolled with 38 treated (20 monotherapy, 18 combination; median 3 prior lines of therapy). The most common treatment-related adverse events (TRAE) were grade 1-2 nausea, fatigue, and diarrhea. No grade 4-5 TRAEs or dose-limiting toxicities were reported. In the monotherapy group, 53% (10/19) of evaluable patients had a best objective response of stable disease. In the combination group, 3 patients had partial responses, for an objective response rate of 20% (3/15) across all doses and 30% (3/10) at TPST-1120 ≥400 mg twice daily. Responses occurred in 2 patients with RCC, both of whom had previously progressed on anti-PD-1 therapy, and 1 patient with late-line CCA., Conclusions: TPST-1120 was well tolerated as monotherapy and in combination with nivolumab and the combination showed preliminary evidence of clinical activity in PD-1 inhibitor refractory and immune compromised cancers., Significance: TPST-1120 is a first-in-class oral inhibitor of PPARα, whose roles in metabolic and immune regulation are implicated in tumor proliferation/survival and inhibition of anticancer immunity. This first-in-human study of TPST-1120 alone and in combination with nivolumab supports proof-of-concept of PPARα inhibition as a target of therapeutic intervention in solid tumors., (© 2024 The Authors; Published by the American Association for Cancer Research.)

The objective of this study is to describe survival outcomes in patients with metastatic melanoma in a real-world setting receiving combination and single-agent immunotherapy outside the clinical trial context. We conducted a retrospective single-institution study of patients with metastatic melanoma in a real-world setting. Survival was calculated using log-rank test. Contingency tables were analyzed using Fisher's Exact test. CD8 + T-cell densities were measured using quantitative immunofluorescence and analyzed using Mann-Whitney U test. The median overall survival (OS) for 132 patients was 45.3 months. Brain metastasis did not confer a higher risk of death relative to liver and/or bone disease (39.53 versus 30.00 months, respectively; P  = 0.687). Anti-PD-1 monotherapy was the most common first-line treatment, received by 49.2% of patients. There was no significant difference in OS between patients receiving single-agent anti-PD-1 and combination anti-PD-1 plus CTLA-4 (39.4 months versus undefined; P  = 0.643). Patients treated with combination therapy were more likely to be alive without progression at the last follow-up than those who received monotherapy (70.4% versus 49.2%; P  = 0.0408). Median OS was 21.8 months after initiation of second-line therapy after anti-PD-1 monotherapy. CD8+ T-cell densities were higher in patients who achieved disease control on first-line immunotherapy ( P  = 0.013). In a real-world setting, patients with metastatic melanoma have excellent survival rates, and treatment benefit can be achieved even after progression on first-line therapy. Combination immunotherapy may produce more favorable long-term outcomes in a real-world setting. High pretreatment CD8+ T-cell infiltration correlates with immunotherapy efficacy., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Dedifferentiation or phenotype switching refers to the transition from a proliferative to an invasive cellular state. We previously identified a 122-gene epigenetic gene signature that classifies primary melanomas as low versus high risk (denoted as Epgn1 or Epgn3). We found that the transcriptomes of the Epgn1 low-risk and Epgn3 high-risk cells are similar to the proliferative and invasive cellular states, respectively. These signatures were further validated in melanoma tumor samples. Examination of the chromatin landscape revealed differential H3K27 acetylation in the Epgn1 low-risk versus Epgn3 high-risk cell lines that corroborated with a differential super-enhancer and enhancer landscape. Melanocytic lineage genes (MITF, its targets and regulators) were associated with super-enhancers in the Epgn1 low-risk state, whereas invasiveness genes were linked with Epgn3 high-risk status. We identified the ITGA3 gene as marked by a super-enhancer element in the Epgn3 invasive cells. Silencing of ITGA3 enhanced invasiveness in both in vitro and in vivo systems, suggesting it as a negative regulator of invasion. In conclusion, we define chromatin landscape changes associated with Epgn1/Epgn3 and phenotype switching during early steps of melanoma progression that regulate transcriptional reprogramming. This super-enhancer and enhancer-driven epigenetic regulatory mechanism resulting in major changes in the transcriptome could be important in future therapeutic targeting efforts.

Background :   Incidence of hepatocellular cancer (HCC) in the Bronx is 61% higher than the rest of New York State. Underserved populations are not well represented in clinical trials of immune checkpoint inhibitors (ICI)., Methods: Demographics were tabulated for 194 patients treated with ICI at the Montefiore-Einstein Comprehensive Cancer Center (MECCC) between 2017 and 2022. Categorical variables were analyzed by Chi-squared test, and survival was analyzed using Kaplan-Meier (KM) curves., Results: MECCC patients were 40.7% Hispanic and 20.6% Black, compared with 3% and 2%, respectively, in the landmark IMbrave 150 study. Median overall survival (mOS) on ICI was 9.0 months, 25.0 months for the 100 (51.5%) favorable-prognosis Child Pugh A (CPA) patients included in HCC clinical trials. Disease control rate (DCR) was 58.5% among 123 evaluable patients per mRECIST 1.1. Baseline liver function, as defined by CP and the Model for End-Stage Liver Disease-Sodium (MELD-Na), correlated with survival (p < 0.001). Hepatitis C Virus (HCV) and alcoholism were over-represented relative to National Cancer Institute (NCI) data (56.2% vs 4.7% and 38.7% vs 8.2%, respectively). HCV treatment correlated with prolonged survival in infected patients (p = 0.0017). AFP decline correlated with response (p = 0.001). Hispanic patients lived longer when clinical variables were controlled for (mOS 52 vs 23 months; p = 0.011)., Conclusion: In an underserved HCC population, ICI yielded a DCR of 58.5% and low rates of severe toxicity. This work highlights ICI efficacy in minority groups, a need for earlier HCC diagnosis and for studies of genetic and environmental factors in Hispanics with HCC., Competing Interests: Yvonne Saenger has received research funding from NextPoint Therapeutics and Regeneron and reports personal fees from Celldex, outside the submitted work. The authors report no other conflicts of interest in this work., (© 2024 Bteich et al.)

Background: Intrarenal complement activation has been implicated in the pathogenesis of tubulointerstitial fibrosis in lupus nephritis (LN) based on prior animal studies. The assembly of the membrane attack complex (MAC) by complement C5b to C9 on the cell membrane leads to cytotoxic pores and cell lysis, while CD59 inhibits MAC formation by preventing C9 from joining the complex. We hypothesize that complement activation and imbalance between complement activation and inhibition, as defined by increased production of individual complement components and uncontrolled MAC activation relative to CD59 inhibition, are associated with interstitial fibrosis and tubular atrophy (IFTA) in LN and correlate with the key mediators of kidney fibrosis- transforming growth factor receptors beta (TGFRβ), platelet-derived growth factor beta (PDGFβ) and platelet-derived growth factor receptor beta (PDGFRβ)., Methods: We included urine samples from 46 adults and pediatric biopsy-proven lupus nephritis patients who underwent clinically indicated kidney biopsies between 2010 and 2019. We compared individual urinary complement components and the urinary C9-to-CD59 ratio between LN patients with moderate/severe IFTA and none/mild IFTA. IFTA was defined as none/mild (<25% of interstitium affected) versus moderate/severe (≥ 25% of interstitium affected). Proteomics analysis was performed using mass spectrometry (Orbitrap Fusion Lumos, Thermo Scientific) and processed by the Proteome Discoverer. Urinary complement proteins enriched in LN patients with moderate/severe IFTA were correlated with serum creatinine, TGFβR1, TGFβR2, PDGFβ, and PDGFRβ., Results: Of the 46 LN patients included in the study, 41 (89.1%) were women, 20 (43.5%) self-identified as Hispanic or Latino, and 26 (56.5%) self-identified as Black or African American. Ten of the 46 (21.7%) LN patients had moderate/severe IFTA on kidney biopsy. LN patients with moderate/severe IFTA had an increased urinary C9-to-CD59 ratio [median 0.91 (0.83-1.05) vs 0.81 (0.76-0.91), p=0.01]. Urinary C3 and CFI levels in LN patients with moderate/severe IFTA were higher compared to those with none/mild IFTA [C3 median (IQR) 24.4(23.5-25.5) vs. 20.2 (18.5-22.2), p= 0.02], [CFI medium (IQR) 28.8 (21.8-30.6) vs. 20.4 (18.5-22.9), p=0.01]. Complement C9, CD59, C3 and CFI correlated with TGFβR1, PDGFβ, and PDGFRβ, while C9, CD59 and C3 correlated with TGFβR2., Conclusion: This study is one of the first to compare the urinary complement profile in LN patients with moderate/severe IFTA and none/mild IFTA in human tissues. This study identified C3, CFI, and C9-to-CD59 ratio as potential markers of tubulointerstitial fibrosis in LN., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier Inc.)

The approval of immunotherapy for stage II-IV melanoma has underscored the need for improved immune-based predictive and prognostic biomarkers. For resectable stage II-III patients, adjuvant immunotherapy has proven clinical benefit, yet many patients experience significant adverse events and may not require therapy. In the metastatic setting, single agent immunotherapy cures many patients but, in some cases, more intensive combination therapies against specific molecular targets are required. Therefore, the establishment of additional biomarkers to determine a patient's disease outcome (i.e., prognostic) or response to treatment (i.e., predictive) is of utmost importance. Multiple methods ranging from gene expression profiling of bulk tissue, to spatial transcriptomics of single cells and artificial intelligence-based image analysis have been utilized to better characterize the immune microenvironment in melanoma to provide novel predictive and prognostic biomarkers. In this review, we will highlight the different techniques currently under investigation for the detection of prognostic and predictive immune biomarkers in melanoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Adeuyan, Gordon, Kenchappa, Bracero, Singh, Espinoza, Geskin and Saenger.)

Purpose: The mortality in patients with MYCN -amplified high-risk neuroblastoma remains greater than 50% despite advances in multimodal therapy. Novel therapies are urgently needed that requires preclinical evaluation in appropriate mice models. Combinatorial treatment with high-dose radiotherapy (HDRT) and immunotherapy has emerged as an effective treatment option in a variety of cancers. Current models of neuroblastoma do not recapitulate the anatomic and immune environment in which multimodal therapies can be effectively tested, and there is a need for an appropriate syngeneic neuroblastoma mice model to study interaction of immunotherapy with host immune cells. Here, we develop a novel syngeneic mouse model of MYCN -amplified neuroblastoma and report the relevance and opportunities of this model to study radiotherapy and immunotherapy., Materials and Methods: A syngeneic allograft tumor model was developed using the murine neuroblastoma cell line 9464D derived a tumor from TH-MYCN transgenic mouse. Tumors were generated by transplanting 1 mm 3 portions of 9464D flank tumors into the left kidney of C57Bl/6 mice. We investigated the effect of combining HDRT with anti-PD1 antibody on tumor growth and tumor microenvironment. HDRT (8 Gy x 3) was delivered by the small animal radiation research platform (SARRP). Tumor growth was monitored by ultrasound. To assess the effect on immune cells tumors sections were co-imuunostained for six biomarkers using the Vectra multispectral imaging platform., Results: Tumor growth was uniform and confined to the kidney in 100% of transplanted tumors. HDRT was largely restricted to the tumor region with minimal scattered out-of-field dose. Combinatorial treatment with HDRT and PD-1 blockade significantly inhibited tumor growth and prolonged mice survival. We observed augmented T-lymphocyte infiltration, especially CD3 + CD8 + lymphocytes, in tumors of mice which received combination treatment., Conclusion: We have developed a novel syngeneic mouse model of MYCN amplified high-risk neuroblastoma. We have utilized this model to show that combining immunotherapy with HDRT inhibits tumor growth and prolongs mice survival., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

Introduction: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium., Methods: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay)., Results: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001)., Conclusion: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: R.A.S. has received fees for professional services from F. Hoffmann-La Roche Ltd, Evaxion, Provectus Biopharmaceuticals Australia, Qbiotics, Novartis, Merck Sharp & Dohme, NeraCare, AMGEN Inc., Bristol-Myers Squibb, Myriad Genetics, GlaxoSmithKline. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: Methods for screening agents earlier in development and strategies for conducting smaller randomized controlled trials (RCTs) are needed., Methods: We retrospectively applied a tumor growth model to estimate the rates of growth of pancreatic cancer using radiographic tumor measurements or serum CA 19-9 values from 3033 patients with stages III-IV PDAC who were enrolled in 8 clinical trials or were included in 2 large real-world data sets., Results: g correlated inversely with OS and was consistently lower in the experimental arms than in the control arms of RCTs. At the individual patient level, g was significantly faster for lesions metastatic to the liver relative to those localized to the pancreas. Regardless of regimen, g increased toward the end of therapy, often by over 3-fold., Conclusions: Growth rates of PDAC can be determined using radiographic tumor measurement and CA 19-9 values. g is inversely associated with OS and can differentiate therapies within the same trial and across trials. g can also be used to characterize changes in the behavior of an individual's PDAC, such as differences in the growth rate of lesions based on metastatic site, and the emergence of chemoresistance. We provide examples of how g can be used to benchmark phase II and III clinical data to a virtual reference arm to inform go/no go decisions and consider novel trial designs to optimize and accelerate drug development., (Published by Oxford University Press 2022.)

It is unclear why some melanomas aggressively metastasize while others remain indolent. Available studies employing multi-omic profiling of melanomas are based on large primary or metastatic tumors. We examine the genomic landscape of early-stage melanomas diagnosed prior to the modern era of immunological treatments. Untreated cases with Stage II/III cutaneous melanoma were identified from institutions throughout the United States, Australia and Spain. FFPE tumor sections were profiled for mutation, methylation and microRNAs. Preliminary results from mutation profiling and clinical pathologic correlates show the distribution of four driver mutation sub-types: 31% BRAF; 18% NRAS; 21% NF1; 26% Triple Wild Type. BRAF mutant tumors had younger age at diagnosis, more associated nevi, more tumor infiltrating lymphocytes, and fewer thick tumors although at generally more advanced stage. NF1 mutant tumors were frequent on the head/neck in older patients with severe solar elastosis, thicker tumors but in earlier stages. Triple Wild Type tumors were predominantly male, frequently on the leg, with more perineural invasion. Mutations in TERT, TP53, CDKN2A and ARID2 were observed often, with TP53 mutations occurring particularly frequently in the NF1 sub-type. The InterMEL study will provide the most extensive multi-omic profiling of early-stage melanoma to date. Initial results demonstrate a nuanced understanding of the mutational and clinicopathological landscape of these early-stage tumors., (© 2022 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Patients with pancreatic ductal adenocarcinoma (PDAC) have a grim prognosis despite complete surgical resection and intense systemic therapies. While immunotherapies have been beneficial with many different types of solid tumors, they have almost uniformly failed in the treatment of PDAC. Understanding how therapies affect the tumor immune microenvironment (TIME) can provide insights for the development of strategies to treat PDAC. We used quantitative multiplexed immunofluorescence (qmIF) quantitative spatial analysis (qSA), and immunogenomic (IG) analysis to analyze formalin-fixed paraffin embedded (FFPE) primary tumor specimens from 44 patients with PDAC including 18 treated with neoadjuvant chemoradiation (CRT) and 26 patients receiving no treatment (NT) and compared them with tissues from 40 treatment-naïve melanoma patients. We find that relative to NT tumors, CD3 + T cell infiltration was increased in CRT treated tumors (p = .0006), including increases in CD3 + CD8 + cytotoxic T cells (CTLs, p = .0079), CD3 + CD4 + FOXP3 - T helper cells (T h , p = .0010), and CD3 + CD4 + FOXP3 + regulatory T cells (Tregs, p = .0089) with no difference in CD68 + macrophages. IG analysis from micro-dissected tissues indicated overexpression of genes involved in antigen presentation, T cell activation, and inflammation in CRT treated tumors. Among treated patients, a higher ratio of Tregs to total T cells was associated with shorter survival time (p = .0121). Despite comparable levels of infiltrating T cells in CRT PDACs to melanoma, PDACs displayed distinct spatial profiles with less T cell clustering as defined by nearest neighbor analysis (p < .001). These findings demonstrate that, while CRT can achieve high T cell densities in PDAC compared to melanoma, phenotype and spatial organization of T cells may limit benefit of T cell infiltration in this immunotherapy-resistant tumor., Competing Interests: YMS has recieved funding from Regeneron. BTF has financial interests in both Regeneron and Thermo Fisher Scientific. GAM is a consultant for CEND Biopharma and Synthekine, and has recieved funding from MERCK, Roche, BioLine, and Regeneron. None of the disclosures listed are related to this work., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Head and neck cell squamous-cell carcinomas (HNSCC) are a group of common cancers typically associated with tobacco use and human papilloma virus infection. Up to half of all cases will suffer a recurrence after primary treatment. As such, new therapies are needed, including therapies which promote the anti-tumor immune response. Prior work has characterized changes in the mutation burden between primary and recurrent tumors; however, little work has characterized the changes in neoantigen evolution. We characterized genomic and neoantigen changes between 23 paired primary and recurrent HNSCC tumors. Twenty-three biopsies from patients originally diagnosed with locally advanced disease were identified from the Washington University tumor bank. Whole exosome sequencing, RNA-seq, and immunohistochemistry was performed on the primary and recurrent tumors. Within these tumors, we identified 6 genes which have predicted neoantigens in 4 or more patients. Interestingly, patients with neoantigens in these shared genes had increased CD3+ CD8+ T cell infiltration and duration of survival with disease. Within HNSCC tumors examined here, there are neoantigens in shared genes by a subset of patients. The presence of neoantigens in these shared genes may promote an anti-tumor immune response which controls tumor progression., Competing Interests: CONFLICTS OF INTEREST Charles Schutt: No disclosures, Hua Sun: No disclosures, Jaya Sarin: No disclosures, Dr. Adkins: Consulting or scientific advisory board support from Pfizer, Merck, Eli Lilly, Celgene (now BMS), Cue Biopharma. Institutional research support from Pfizer Eli Lilly, Merck, Celgene/BMS, Novartis, AstraZeneca, Atara Bio, Blueprint Medicine, Celldex, Enzychem, Kura, Exelixis, Innate, Sensei, and Matrix Biomed. Yvonne Saenger: Research funding from Amgen and Regeneron. Equity on a biomarker startup, Wasaba. Jessica Ley: No disclosures. Matthew Ingham: Consulting or Advisory role: Daiichi Sanky; Research Funding: Mirati Therapeutics, Apexigen, PTC Therapeutics; Travel, Accommodations, Expenses: Genetech, Mirati Therapeutics. Li Ding: No disclosures. Brian Van Tine: Grants from Merck, grants and personal fees from Pfizer, grants from Tracon, grants, personal fees and other from GSK, personal fees from Polaris, personal fees from Lilly, personal fees from Caris Life Sciences, personal fees from Novartis, personal fees from CytRX, personal fees from Plexxikon, personal fees from Epizyme, personal fees from Daiihi Sankyo, personal fees from Adaptimmune, personal fees from Immune Design, personal fees from Bayer, personal fees from Cytokinetics, personal fees from Deciphera. In addition, Dr. Van Tine has a patent on the use of ME1 as biomarker and ALEXT3102 issued., (Copyright: © 2021 Schutt et al.)

Accurate prognostic biomarkers in early-stage melanoma are urgently needed to stratify patients for clinical trials of adjuvant therapy. We applied a previously developed open source deep learning algorithm to detect tumor-infiltrating lymphocytes (TILs) in hematoxylin and eosin (H&E) images of early-stage melanomas. We tested whether automated digital (TIL) analysis (ADTA) improved accuracy of prediction of disease specific survival (DSS) based on current pathology standards. ADTA was applied to a training cohort (n = 80) and a cutoff value was defined based on a Receiver Operating Curve. ADTA was then applied to a validation cohort (n = 145) and the previously determined cutoff value was used to stratify high and low risk patients, as demonstrated by Kaplan-Meier analysis (p ≤ 0.001). Multivariable Cox proportional hazards analysis was performed using ADTA, depth, and ulceration as co-variables and showed that ADTA contributed to DSS prediction (HR: 4.18, CI 1.51-11.58, p = 0.006). ADTA provides an effective and attainable assessment of TILs and should be further evaluated in larger studies for inclusion in staging algorithms.

Background: The study of immune surveillance in the tumour microenvironment is leading to the development of new biomarkers and therapies. The present research focuses on the expression of CD5 and CD6 -two lymphocyte surface markers involved in the fine tuning of TCR signaling-as potential prognostic biomarkers in resectable stages of non-small cell lung cancer (NSCLC)., Methods: CD5 and CD6 gene expression was analysed by reverse transcription quantitative polymerase chain reaction (RTqPCR) in 186 paired fresh frozen tumour and normal tissue samples of resected NSCLC., Results: Patients with higher CD5 expression had significantly increased overall survival (OS, 49.63 vs . 99.90 months, P=0.013). CD5 expression levels were correlated to CD4 infiltration and expression levels, and survival analysis showed that patients with a higher CD5 / CD4 + ratio had significantly improved prognosis. Multivariate analysis established C D5 expression as an independent prognostic biomarker for OS in early stages of NSCLC (HR=0.554; 95% CI, 0.360-0.853; P=0.007). Further survival analysis of NSCLC cases (n=97) from The Cancer Genome Atlas (TCGA) database, confirmed the prognostic value of both CD5 and CD6 expression¸ although CD6 expression alone did not reach significant prognostic value in our NSCLC training cohort., Conclusions: Our data support further studies on CD5 and CD6 as novel prognostic markers in resectable NSCLC and other cancer types (i.e., melanoma), as well as a role for these receptors in immune surveillance., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr-19-445). RR serves as an unpaid editorial board member of Translational Lung Cancer Research from Jun 2019 to May 2022. The other authors have no conflicts of interest to declare., (2020 Translational Lung Cancer Research. All rights reserved.)

Response to talimogene laherparepvec (T-Vec) is difficult to assess as pigmented macrophages that have ingested melanoma cells ('melanophages') persist after injection, mimicking melanoma. We used quantitative immunofluorescence (qIF) to (1) distinguish melanophages from melanoma in biopsies from two patients treated with T-Vec and (2) evaluate the tumor microenvironment pretreatment and posttreatment. Tissues were stained with 4',6-diamidino-2-phenylindole, cluster of differentiation (CD) 3, CD8, CD68, human leukocyte antigen-DR isotype (HLA-DR), and SRY-Box Transcription Factor 10 (SOX10), and multispectral images were analyzed. Post-T-Vec samples showed melanophages with cytoplasmic costaining of CD68, SOX10, and HLA-DR, without nuclear SOX10 expression. qIF revealed a dense immune infiltrate of CD3, CD8, and CD68 cells in post-T-Vec samples. Melanophages from tumors post-T-Vec stain the nuclear melanoma marker SOX10 in their cytoplasms as compared to melanoma cells that stain nuclear SOX10. This novel finding highlights the phagocytosis of melanoma cell components by macrophages after treatment with T-Vec. qIF may assist pathologists in determining whether lesions treated with immunotherapy contain residual viable melanoma.

Background: The PI3K/Akt/mTOR pathway in part impacts tumorigenesis through modulation of host immune activity. To assess the effects of Akt inhibition on the tumor micro-environment (TME), we analyzed tumor tissue from patients with operable hormone receptor positive, HER2 negative breast cancer (BC) treated on a presurgical trial with the Akt inhibitor MK-2206. Methods: Quantitative multiplex immunofluorescence (qmIF) was performed using CD3, CD8, CD4, FOXP3, CD68, and pancytokeratin on biopsy and surgical specimens of MK-2206 and untreated, control patients. nanoString was performed on surgical specimens to assess mRNA expression from MK-2206-treated vs. control patients. Results: Increased CD3+CD8+ density was observed in post vs. pre-treatment tissue in the MK-2206-treated vs. control patients (87 vs. 0.2%, p < 0.05). MK-2206 was associated with greater expression of interferon signaling genes (e.g., IFI6 , p < 0.05) and lower expression of myeloid genes ( CD163 , p < 0.05) on differential expression and gene set enrichment analyses. Greater expression of pro-apoptotic genes (e.g., BAD ) were associated with MK-2206 treatment ( p < 0.05). Conclusion: Akt inhibition in operable BC was associated with a favorable immune profile in the TME, including increased CD3+CD8+ density and greater expression of interferon genes. Additional studies are warranted, as this may provide rationale for combining Akt inhibition with immunotherapy., (Copyright © 2020 Marks, Gartrell, El Asmar, Boboila, Hart, Lu, Pan, Yu, Hibshoosh, Guo, Andreopoulou, Wiechmann, Crew, Sparano, Hershman, Connolly, Saenger and Kalinsky.)

Background: Immune checkpoint blockade has emerged as a highly effective treatment for patients with metastatic melanoma and cutaneous squamous cell carcinoma. Nivolumab blocks the interactions between programmed cell death protein 1 and programmed death ligand 1 allowing for activation of a latent immune response against the malignancy. Ipilimumab binds to and blocks cytotoxic T-lymphocyte-associated protein 4, alleviating the negative regulation of T-cell activation that is mediated by that checkpoint. Combination therapy with nivolumab and ipilimumab is associated with longer overall survival at 5 years compared with nivolumab monotherapy. Solid organ transplant recipients have a significantly higher risk of malignancies compared with the general population. There is limited data surrounding the efficacy of combination immunotherapy in solid organ transplant recipients, as these patients were excluded from seminal trials due to risk of organ rejection., Case Presentations: Here we present four cases of combination immunotherapy in kidney transplant recipients. Three patients had metastatic melanoma, and one patient had metastatic cutaneous squamous cell carcinoma. Two patients had radiographic responses from immunotherapy, one patient had stable disease, and one patient had disease progression. Only one patient had biopsy-proven rejection. At last follow-up, three patients had functioning grafts, though one required hemodialysis after treatment, and one patient succumbed to disease, but graft function remained intact throughout her course., Conclusions: These cases describe the use of ipilimumab and nivolumab combination immunotherapy for cutaneous malignancies in kidney transplant recipients. They highlight the potential to preserve kidney graft function while effectively treating the disease., Trial Registration Number: NCT03816332., Competing Interests: Competing interests: RC is a consultant for Astra Zeneca, BMS, Castle Biosciences, Foundation Medicine, Immunocore, Incyte, Merck, Novartis, Roche, Compugen, I-Mab, PureTech Health, Sanofi Genzyme, and Sorrento Therapeutics. He is on advisory boards for Aura Biosciences, Chimeron, and Rgenix., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Investigate the ability of F-fluorothymidine (FLT) PET combined with CT at 6 weeks to predict treatment response at 12 weeks after treatment with pembrolizumab., Methods: Five patients with unresectable stage IV melanoma were included in this single-institution pilot study. Patients underwent FLT-PET/CT (baseline and 6 weeks) and CT (baseline and 12 weeks). FLT-PET/CT response and CT response were assessed using PET Response Criteria in Solid Tumors and immune Response Evaluation Criteria in Solid Tumors, respectively. Patients were categorized as responders (complete response, partial response) and nonresponders (stable disease, progressive disease). Agreement between 6-week FLT-PET/CT and 12-week CT was calculated using Cohen kappa's agreement. Eight baseline FLT-PET/CT parameters were extracted: SUVmax, SUVpeak, SUVSD, SUVmean, proliferative tumor volume, total lesion proliferation, bone marrow-to-liver SUVmax ratio, and spleen-to-liver SUVmax ratio. Eight delta-parameters were extracted at 6 weeks by calculating variation in FLT uptake as percentage change from baseline., Results: Agreement between 6-week FLT-PET/CT and 12-week CT was kappa = 0.615, P = 0.025. Three of 5 patients were categorized as responders on CT by immune Response Evaluation Criteria in Solid Tumors. At baseline, responders had a lower mean proliferative tumor volume and a higher bone marrow-to-liver SUVmax ratio. At 6 weeks, responders demonstrated a decrease in tumor volume and tumor proliferation., Conclusions: Our study illustrates the potential for FLT-PET/CT as an early predictor of response for patients with metastatic melanoma on anti-PD1 immunotherapy. Larger studies are indicated to confirm these findings.

Opinion Statement: There is an unmet need for additional treatments for metastatic melanoma, besides anti-PD1 antibodies which are FDA approved for adjuvant therapy for stage III or resected stage IV melanoma. Talimogene laherparepvec (T-VEC) is the first and only FDA-approved oncolytic virus for the treatment of melanoma. New viral vectors including coxsackieviruses, HF-10, adenovirus, reovirus, echovirus, and newcastle disease virus are currently under active development and investigation with varying degrees of efficacy in targeting melanoma. The use of T-VEC as a neoadjuvant therapy is emerging, but more data is needed at this point. T-VEC has also shown promise for use in combination therapy with ipilimumab, as T-VEC plus ipilimumab has a significantly higher objective response compared to ipilimumab alone. Data comparing T-VEC in combination with PD-1 checkpoint inhibitors is awaited, and a phase III trial is underway. It is likely that oncolytic viruses will have long-term application in the treatment of melanoma and that T-VEC in particular will continue to have a role in the treatment of patients with readily accessible cutaneous lesions both for local control and synergistic induction of antitumor immunity as part of combination therapies.

Patients with resected stage II-III melanoma have approximately a 35% chance of death from their disease. A deeper understanding of the tumor immune microenvironment (TIME) is required to stratify patients and identify factors leading to therapy resistance. We previously identified that the melanoma immune profile (MIP), an IFN-based gene signature, and the ratio of CD8 + cytotoxic T lymphocytes (CTL) to CD68 + macrophages both predict disease-specific survival (DSS). Here, we compared primary with metastatic tumors and found that the nuclei of tumor cells were significantly larger in metastases. The CTL/macrophage ratio was significantly different between primary tumors without distant metastatic recurrence (DMR) and metastases. Patients without DMR had higher degrees of clustering between tumor cells and CTLs, and between tumor cells and HLA-DR + macrophages, but not HLA-DR - macrophages. The HLA-DR - subset coexpressed CD163 + CSF1R + at higher levels than CD68 + HLA-DR + macrophages, consistent with an M2 phenotype. Finally, combined transcriptomic and multiplex data revealed that densities of CD8 and M1 macrophages correlated with their respective cell phenotype signatures. Combination of the MIP signature with the CTL/macrophage ratio stratified patients into three risk groups that were predictive of DSS, highlighting the potential use of combination biomarkers for adjuvant therapy. SIGNIFICANCE: These findings provide a deeper understanding of the tumor immune microenvironment by combining multiple modalities to stratify patients into risk groups, a critical step to improving the management of patients with melanoma., (©2020 American Association for Cancer Research.)

Purpose: Biomarkers for disease-specific survival (DSS) in early-stage melanoma are needed to select patients for adjuvant immunotherapy and accelerate clinical trial design. We present a pathology-based computational method using a deep neural network architecture for DSS prediction., Experimental Design: The model was trained on 108 patients from four institutions and tested on 104 patients from Yale School of Medicine (YSM, New Haven, CT). A receiver operating characteristic (ROC) curve was generated on the basis of vote aggregation of individual image sequences, an optimized cutoff was selected, and the computational model was tested on a third independent population of 51 patients from Geisinger Health Systems (GHS)., Results: Area under the curve (AUC) in the YSM patients was 0.905 ( P < 0.0001). AUC in the GHS patients was 0.880 ( P < 0.0001). Using the cutoff selected in the YSM cohort, the computational model predicted DSS in the GHS cohort based on Kaplan-Meier (KM) analysis ( P < 0.0001)., Conclusions: The novel method presented is applicable to digital images, obviating the need for sample shipment and manipulation and representing a practical advance over current genetic and IHC-based methods., (©2019 American Association for Cancer Research.)

Non-recirculating tissue-resident memory T cells (TRMs) are the predominant T cell subset in diverse tissue sites, where they mediate protective immune responses in situ. Here, we reveal a role for TRM in maintaining immune homeostasis in the human pancreas through interactions with resident macrophages and the PD-1/PD-L1 inhibitory pathway. Using tissues obtained from organ donors, we identify that pancreas T cells comprise CD8 + PD-1 hi TRMs, which are phenotypically, functionally, and transcriptionally distinct compared to TRMs in neighboring jejunum and lymph node sites. Pancreas TRMs cluster with resident macrophages throughout the exocrine areas; TRM effector functions are enhanced by macrophage-derived co-stimulation and attenuated by the PD-1/PD-L1 pathways. Conversely, in samples from chronic pancreatitis, TRMs exhibit reduced PD-1 expression and reduced interactions with macrophages. These findings suggest important roles for PD-1 and TRM-macrophage interactions in controlling tissue homeostasis and immune dysfunctions underlying inflammatory disease, with important implications for PD-1-based immunotherapies., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Assessment of tumor infiltrating lymphocytes (TILs) as a prognostic variable in melanoma has not seen broad adoption due to lack of standardization. Automation could represent a solution. Here, using open source software, we build an algorithm for image-based automated assessment of TILs on hematoxylin-eosin stained sections in melanoma. Using a retrospective collection of 641 melanoma patients comprising four independent cohorts; one training set (N = 227) and three validation cohorts (N = 137, N = 201, N = 76) from 2 institutions, we show that the automated TIL scoring algorithm separates patients into favorable and poor prognosis cohorts, where higher TILs scores were associated with favorable prognosis. In multivariable analyses, automated TIL scores show an independent association with disease-specific overall survival. Therefore, the open source, automated TIL scoring is an independent prognostic marker in melanoma. With further study, we believe that this algorithm could be useful to define a subset of patients that could potentially be spared immunotherapy.

Immune checkpoint inhibitors have been successful across several tumor types; however, their efficacy has been uncommon and unpredictable in glioblastomas (GBM), where <10% of patients show long-term responses. To understand the molecular determinants of immunotherapeutic response in GBM, we longitudinally profiled 66 patients, including 17 long-term responders, during standard therapy and after treatment with PD-1 inhibitors (nivolumab or pembrolizumab). Genomic and transcriptomic analysis revealed a significant enrichment of PTEN mutations associated with immunosuppressive expression signatures in non-responders, and an enrichment of MAPK pathway alterations (PTPN11, BRAF) in responders. Responsive tumors were also associated with branched patterns of evolution from the elimination of neoepitopes as well as with differences in T cell clonal diversity and tumor microenvironment profiles. Our study shows that clinical response to anti-PD-1 immunotherapy in GBM is associated with specific molecular alterations, immune expression signatures, and immune infiltration that reflect the tumor's clonal evolution during treatment.

A correction was made to a sentence in the original article to provide additional clarification in the "Other Oncolytic Viruses" section.

Purpose of Review: Oncolytic virotherapy is a new approach to the treatment of cancer and its success in the treatment of melanoma represents a breakthrough in cancer therapeutics. This paper provides a review of the current literature on the use of oncolytic viruses (OVs) in the treatment of melanoma., Recent Findings: Talimogene laherparepvec (T-VEC) is the first OV approved for the treatment of melanoma and presents new challenges as it enters the clinical setting. Several other OVs are at various stages of clinical and pre-clinical development for the treatment of melanoma. Reports from phase Ib-III clinical trials combining T-VEC with checkpoint blockade are encouraging and demonstrate potential added benefit of combination immunotherapy. OVs have recently emerged as a standard treatment option for patients with advanced melanoma. Several OVs and therapeutic combinations are in development. Immunooncolytic virotherapy combined with immune checkpoint inhibitors is promising for the treatment of advanced melanoma.

Background: Immune checkpoint inhibitors (ICIs) have changed the clinical management of melanoma. However, not all patients respond, and current biomarkers including PD-L1 and mutational burden show incomplete predictive performance. The clinical validity and utility of complex biomarkers have not been studied in melanoma., Methods: Cutaneous metastatic melanoma patients at eight institutions were evaluated for PD-L1 expression, CD8 + T-cell infiltration pattern, mutational burden, and 394 immune transcript expression. PD-L1 IHC and mutational burden were assessed for association with overall survival (OS) in 94 patients treated prior to ICI approval by the FDA (historical-controls), and in 137 patients treated with ICIs. Unsupervised analysis revealed distinct immune-clusters with separate response rates. This comprehensive immune profiling data were then integrated to generate a continuous Response Score (RS) based upon response criteria (RECIST v.1.1). RS was developed using a single institution training cohort (n = 48) and subsequently tested in a separate eight institution validation cohort (n = 29) to mimic a real-world clinical scenario., Results: PD-L1 positivity ≥1% correlated with response and OS in ICI-treated patients, but demonstrated limited predictive performance. High mutational burden was associated with response in ICI-treated patients, but not with OS. Comprehensive immune profiling using RS demonstrated higher sensitivity (72.2%) compared to PD-L1 IHC (34.25%) and tumor mutational burden (32.5%), but with similar specificity., Conclusions: In this study, the response score derived from comprehensive immune profiling in a limited melanoma cohort showed improved predictive performance as compared to PD-L1 IHC and tumor mutational burden.

Introduction: Ulcerated melanomas may have a unique biology and microenvironment. We test whether markers of immune infiltration correlate with clinical outcome in ulcerated compared to non-ulcerated primary melanoma tumors., Methods: Sixty-two stage II-III cutaneous melanomas, 32 ulcerated and 30 non-ulcerated, were analyzed for tumor-infiltrating lymphocytes (TILs). Immunohistochemistry (IHC) was performed for CD2, a marker previously shown to correlate with overall survival (OS) and recurrence-free survival (RFS) in this patient population. IHC using antibody, VE1, to BRAF V600E was also performed on a subset of 41 tumors to assess the relationship of BRAF mutation to immune markers., Results: We found, using Cox regression models, that the presence of TILs was associated with improved OS (p = 0.034) and RFS (p = 0.002) in ulcerated melanoma tumors, but not in non-ulcerated melanoma (p = 0.632, 0.416). CD2 expression also was correlated with improved OS (p = 0.021) and RFS (p = 0.001) in ulcerated melanoma, but no relationship was seen in non-ulcerated melanoma (p = 0.427, 0.682). In this small population, BRAF status did not correlate with TILs or CD2+ count., Conclusion: Our data show that immune markers including TILs and CD2 count correlate more closely with survival in ulcerated melanomas than that in non-ulcerated melanomas. We propose that immune biomarkers may be particularly relevant to ulcerated, as compared to non-ulcerated, melanomas and that this merits study in larger populations.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a negative regulator of T-cell mediated immune responses and is the target of new anti-tumor immunotherapy strategies. Ipilimumab is a fully human, antagonistic monoclonal antibody directed against CTLA-4. Results from preclinical and early clinical trials support current phase II/III testing of ipilimumab as first- and second-line therapy for metastatic melanoma. Ipilimumab promotes durable objective responses and/or stable disease in patients with metastatic melanoma. Adverse events are medically manageable, largely immune-related, and presumably linked to the drug's mechanism of action. As more patients are treated with ipilimumab, it is becoming clear that the kinetics of responses are heterogeneous and significantly different from those of chemotherapy and other immunotherapy. Though objective response or stable disease is observed within 'conventional' time frames, responses have been observed weeks to months after therapy initiation. Response or stable disease may be preceded by apparent early disease progression, or may occur simultaneously with different progressing lesions within the same patient (a 'mixed' response). It is likely that the unique kinetics of response is a result of the time required to enhance and maintain an anti-tumor immune response to ipilimumab therapy. Consequently, patients may benefit from continued ipilimumab treatment through clinically known relevant disease progression or non-response during the full induction dosing schedule (12 weeks), without additional therapies. Understanding the kinetics of response to ipilimumab will help clinicians to manage patients who may benefit from treatment. In this article, several cases that illustrate the kinetics of response to ipilimumab are discussed.

The structure recognized by regulatory T cells that enables them to discriminate self from nonself in the periphery is one of the central issues of regulatory T cell biology. A link between immunoregulation and self-nonself discrimination has emerged from experiments showing that Qa-1-restricted CD8(+) T cells selectively down-regulate target T cells activated by the intermediate avidity of their own T cell antigen receptor-ligand interactions. Because the peripheral self-reactive T cell repertoire is devoid of high-avidity T cells compared with the foreign-reactive repertoire, as a result of thymic negative selection, the selective down-regulation of intermediate but not high-avidity T cells enables the immune system to suppress autoimmunity without damaging the ongoing immune response to foreign pathogens. However, the molecular mechanism delineating how avidity of T cell activation is perceived by the regulatory T cells has not been elucidated. Here we show that a heat shock peptide (Hsp60sp), coupled with the MHC class Ib molecule Qa-1, is a surrogate target structure that is preferentially expressed at a higher level on the intermediate avidity T cells and specifically recognized by the Qa-1-restricted CD8(+) T cells. The biological significance of this observation was confirmed by the ability of Hsp60sp-loaded relevant dendritic cells to induce a Qa-1-restricted CD8(+) T cell-mediated protection from autoimmune encephalopathy in the experimental allergic encephalomyelitis model. Thus, perceiving the avidity of T cell activation can be translated into peripheral T cell regulation to discriminate self from nonself in the periphery to maintain self-tolerance.